Hydrothermal liquefaction aqueous phase mycoremediation to increase inorganic nitrogen availability

Hydrothermal liquefaction aqueous phase (HTL-AP) is a waste product from a thermochemical process where wet biomass is converted into biocrude oil. This nutrient-rich wastewater may be repurposed to benefit society by assisting crop growth after adequate treatment to increase inorganic nitrogen, especially NO3−. This study aims to increase HTL-AP inorganic nitrogen, specifically NH3/NH4+ and NO3−, through fungal remediation for further use in hydroponic systems. Trametes versicolor, a white-rot fungus known for degrading a range of organic pollutants, was used to treat a diluted (5 %) HTL-AP for 9 days. No fungal growth was observed, but T. versicolor activity was suspected by laccase activity throughout cultivation time. NO3−-N and NH3/NH4+-N increased by 17 and 8 times after three days of fungal treatment, which was chosen as the appropriate time for HTL-AP fungal treatment as it resulted in the highest concentration of NO3−-N. The addition of nitrifying bacteria to the fungal treatment resulted in a twofold increase in NO3−-N concentration compared to the fungal treatment alone, indicating an enhancement in treatment efficacy. COD decreased by 51.33 % after 24 h, which may be related to the fungus’ capacity to reduce the concentration of organics in the wastewater; nonetheless, COD increased in the following days, which may be related to the release of fungal byproducts. T. versicolor shows promise as a potential candidate for increasing inorganic nitrogen in HTL-AP. However, future studies should primarily address HTL-AP toxicity, reducing NH3/NH4+-N while increasing NO3−-N, and hydroponics crop production after fungal treatment.


Introduction
Fossil fuels' extensive use has introduced environmental problems around the world, directing attention to alternative fuels [1,2].For instance, the hydrothermal liquefaction (HTL) of biomass has drawn attention as an alternative method for producing biocrude oil, which can be further upgraded to transportation biofuel [1,3].This process can use a range of biomasses as feedstock, including waste products such as food waste [4].The Office of Resource Conservation and Recovery [5] estimated the generation of around 103 million tons of food waste in the United States in 2018.Hence, HTL is a potential solution to deal with two issues modern society faces: fossil fuels and food waste generation.However, a complex and potentially toxic liquid stream called hydrothermal liquefaction aqueous Abbreviations: HTL-AP, Hydrothermal liquefaction aqueous phase; NO 3 -N, nitrate-nitrogen; NO 3 − , nitrate; NH 3 /NH 4 + -N, ammonia/ammoniumnitrogen; NH 3 /NH 4 + , ammonia/ammonium; NO 2 − , nitrite; COD, chemical oxygen demand.
phase (HTL-AP) is generated as a byproduct that needs to be treated before discharged into the environment [6].HTL-AP may contain heavy metals and has a high concentration of organics, including nitrogen-containing organics and aromatic compounds, that impose challenges on its treatment or further use [4].Alternatively, HTL-AP has the potential for reuse in hydroponic systems after adequate treatment, as it is free from pathogens due to the high temperature and pressure conditions of the HTL process, yet contains nutrients that can support crop growth [7].Nonetheless, most of the nitrogen in HTL-AP from swine manure HTL is in organic form and not readily accessible to plants, which uptake nitrogen as ions (e.g., NH 4 + and NO 3 − ) [8,9].This underscores the need for new studies that seek to increase inorganic nitrogen availability in HTL-AP to increase the overall value of the HTL process.Biological treatment of HTL-AP has been explored and possesses several advantages, such as its eco-friendly nature, costeffectiveness, lower energy consumption compared to traditional methods, and ability to reduce toxicity, coupled with the potential to produce value-added compounds [4,10].He et al. [11] explored different bacteria strains from Rhodococci sp. to produce lipid-rich bacterial biomass from algae or woody substrates when grown in HTL-AP.Si et al. [12] sought to produce biohythane from HTL-AP using two-stage fermentation, where hydrogen gas and organic acids were produced in the first step by substrate hydrolyzation, and the organic acids transformation further generated methane.Chen et al. [13] assessed algae biomass production from HTL-AP.Similarly, Yang et al. [14] tested algae biomass cultivation with HTL-AP that was pre-treated using anaerobic digestion as a detoxification step and for methane production.Cordova et al. [15] used engineered yeast strains to produce the value-added chemicals itaconic acid and triacetic acid lactone using HTL-AP as a complementary nutrient source.Bioelectrochemical systems were also explored for HTL-AP treatment for electricity and hydrogen generation [16,17].All the mentioned studies indicate that microorganisms can grow using HTL-AP as a nutrient source and produce value-added compounds.However, none of them focused on increasing inorganic nitrogen in this wastewater.
Emerging as an alternative biological treatment, fungal treatment of HTL-AP may increase inorganic nitrogen availability while also reducing its toxicity, which is mainly related to the presence of heavy metals and organic nitrogen molecules, aromatic or not [4,18].Fungal treatment, also called mycoremediation, is an eco-friendly approach where fungal species degrade, remove, or convert contaminants from wastewater through different mechanisms [19].Fungi can uptake small organic nitrogen compounds and release their excess as NH 3 /NH 4 + through ammonification [20,21].Additionally, fungal mycelium has shown a significant sorption capacity to remove heavy metals and organic molecules from liquid streams, and some fungal species, specifically from the white-rot fungus group, can excrete enzymes (e.g., laccase) that have the capacity to degrade a range of organic nitrogen pollutants [22][23][24][25][26].These microorganisms tend to excrete these enzymes to transform complex nutrients into simpler ones, making them easier to utilize [27].Therefore, mycoremediation may be a potential alternative to increase inorganic nitrogen while reducing HTL-AP toxicity, aiming for future use of this waste stream as fertilizer in hydroponic systems.Among all fungal species, white-rot fungi have stood out as a promising fungal group for pollutant removal due to their enzymatic machinery and stability under adverse conditions [28,29].They have been explored for the mycoremediation of a range of recalcitrant organic pollutants, such as dyes, pharmaceutical compounds, phenols, and pesticides [28].Trametes versicolor is the most widely investigated fungus from this group and has been reported to treat wastewater containing complex organic pollutants and heavy metals [23,30,31].While T. versicolor has been explored for other wastewaters and in combination with other biological treatments [32], there are no studies addressing how long the fungus should be cultivated in HTL-AP.In this context, to the best of the authors' knowledge, this is the first study addressing T. versicolor optimal cultivation time when used as the sole source of treatment to increase inorganic nitrogen in HTL-AP.Therefore, the contribution of this study is to investigate the increase of inorganic nitrogen in HTL-AP by cultivating the fungus T. versicolor in this wastewater, thereby facilitating the potential utilization of this waste stream as a fertilizer in hydroponic systems.Several parameters (NH 3 /NH 4 + -nitrogen, NO 3 − -nitrogen, COD, biomass dry weight, and laccase activity) were evaluated across 9 days of fungal cultivation to determine the effect of mycoremediation on HTL-AP for increasing inorganic nitrogen concentration, as well as the time needed for maximum treatment effectiveness.Additionally, fungal treatment along with nitrifying bacteria was explored to evaluate the increase in NO 3 − concentration.

Hydrothermal liquefaction aqueous phase (HTL-AP)
HTL-AP was provided by the Environment-Enhancing Energy (E2-E) Laboratory (University of Illinois at Urbana-Champaign) from the hydrothermal liquefaction of food waste (at 280 • C, 30 min retention time).The raw HTL-AP was stored at 4 • C until used and its biochemical properties are described in Table 1.For these experiments, HTL-AP was diluted to 5 % in DI water and stored at 4 • C. The 5 % dilution was chosen based on a previous study conducted by Jesse et al. [8], which evaluated hydroponic lettuce production using 2.5 % HTL-AP from swine manure.While some crop growth was observed when using the 2.5 % HTL-AP, the growth was less than the crops receiving a standard hydroponic fertilizer.Therefore, the concentration in this study was doubled to utilize a higher percentage of HTL-AP but still low enough for future evaluation in hydroponic leafy green crop production.

Fungal strain
Trametes versicolor was kindly donated by the Miller Mycology Lab -Illinois Natural History Survey (University of Illinois at Urbana-Champaign).The fungal species was routinely cultivated in sterile potato dextrose agar (PDA) medium (15 psi, 121 • C, 15 min) at 28 • C for 5-7 days and stored at 4 • C until use.

Mycelial suspension
The mycelial suspension was prepared according to Blánquez et al. [33] with some modifications.Briefly, a 500 mL Erlenmeyer containing 150 mL sterile malt extract medium (ME, 2 % w/v, 15 psi, 121 • C, 15 min) was inoculated with 4 mycelial plugs (1 cm diameter) from the fungus growing region on PDA.The flask was sealed with a gauze filter followed by a KC100 sterilization wrap and incubated for 4 days at 22 ± 1 • C and 135 rpm.The resulting thick mycelial plugs were washed twice with sterile DI water (15 psi, 121 • C, 15 min), and immersed in sterile 0.85 % NaCl (15 psi, 121 • C, 15 min).Lastly, the fungal plugs in saline solution were ground and mixed using a bead beater homogenizer by the addition of 6 matrix M beads (MP biomedicals; Irvin, California, U.S.A.; 60 s at 6.5 m/s).The mycelial suspension was stored at 4 • C until use.

Pellets production
Fungal pellets were produced following modified methods from Blánquez et al. [33].Briefly, 1 mL of mycelial suspension was added to a 1 L flask containing 250 mL of sterile malt extract medium (2 % w/v).The samples were incubated at 135 rpm and 22 ± 1 • C for 6 days.The pellets were transferred aseptically to centrifuge tubes (50 mL), washed twice with sterile DI water, and stored at 4 • C with the addition of sterile saline solution (0.85 % NaCl) until use.

Wastewater time-experiment
The wastewater time-experiment was carried out in triplicates in 250 mL flasks containing 50 mL of 5 % sterile HTL-AP (v/v, 121 • C, 15 min) inoculated with 6.4 % (w/v) wet fungal pellets.The HTL-AP concentration was selected based on findings from prior research.Jesse et al. ( 2019) successfully demonstrated lettuce growth in treated 2.5 % HTL-AP [8].Here, 5 % is proposed to increase the HTL-AP concentration that can be used to grow crops successfully.Positive controls (duplicates) were prepared with 2 % (w/v) malt extract broth and negative controls (triplicates) with 5 % HTL-AP without inoculation.Negative controls were used for comparison to the treated samples.The samples were then incubated (135 rpm, 28 • C) and collected according to the time scheduled (4,24,46,72,96,118,168, and 214 h).While negative controls were obtained for each time data point, positive controls were analyzed after 214 h of cultivation time.After the incubation period, the samples were filtered (Whatman 42) and stored at 4 • C until analyzed.For dry biomass, fungal pellets were dried at 70 • C for 2 h and 10 min.Lastly, HTL-AP pH was recorded before and after fungal cultivation.

Wastewater nitrifying experiment
The wastewater nitrifying experiments were carried out in triplicates in 250 mL flasks containing 50 mL of 5 % sterile HTL-AP (v/v, 121 • C, 15 min).Flasks were aseptically inoculated with either 6.4 % (w/v) wet fungal pellets (Tv-5HTL-AP) or 190 μL ATM Aquarium Products Colony Nitrifying Bacteria (ATM Aquarium Products, Las Vegas, NV, USA) containing the bacteria Nitrosomonas and Nitrobacter (B-5HTL-AP), or the combination of both (B + Tv-5HTL-AP).Negative controls were prepared and labeled as 5HTL-AP.The samples were incubated for 3 days at 28 • C and 135 rpm.Following incubation, the samples were filtered (0.45 μm) and stored at 4 • C until analyzed.

Wastewater characterization 2.7.1. Nutrients analysis
Measurements for ammonia/ammonium-nitrogen (NH 3 /NH 4 + -N), nitrate-nitrogen (NO 3 − -N), and chemical oxygen demand (COD) were performed according to Hach methodologies 8038, 8039, and 8000, respectively.For each measurement, at least triplicates were analyzed.Additionally, for NO 3 − -N and COD measurements, only two negative controls were randomly selected for analysis.NO 3 −nitrogen and NH 3 /NH 4 + -nitrogen readings were performed using Hach DR/2010 spectrophotometer (Loveland, Colorado, U.S.A.), while Hach DR/3900 (Loveland, Colorado, U.S.A.) was used for COD.The results are presented as the average of the readings with their respective standard deviations.

Statistical analysis
An ANOVA was utilized to investigate the effects of time of incubation and fungal treatment on COD and NO 3 − -N.The objective was to determine if there were significant differences in mean levels of COD and NO 3 − -N across various time points and fungal treatments.
Furthermore, a Kruskal-Wallis test was employed to evaluate the effects of time of incubation and fungal treatment on NH 3 /NH 4

+
-N.This non-parametric test was preferred as the distribution of NH 3 /NH 4 + -N levels did not meet the normality assumptions needed for ANOVA.

Wastewater time-experiment
Changes in pH, NH 3 /NH 4 + -N, NO 3 − -N, COD, dry weight, and laccase activity were monitored over time to assess the impact of fungal cultivation on wastewater treatment.Results for dry weight and laccase activity are presented in Fig. 1 (a).Fungal dry biomass slightly decreased over time (Fig. 1, a).Cruz-Morató et al. [35] reported a similar behavior and explained it as the lack of nutrients in the medium, which may cause mycelium lysis and a further decrease in biomass.While a low dry mass was observed in HTL-AP inoculated samples (<0.14 g) during the cultivation period, the positive control dry mass after 214 h resulted in 2.05 ± 0.03 g of dry biomass, implying the activity of the fungal inoculum employed in this experiment.Enzyme activity by T. versicolor increased over time (Fig. 1, a), suggesting fungal activity during wastewater cultivation [30].No activity was detected in the negative controls, suggesting that laccase activity was solely attributed to the presence of T. versicolor, as all samples were autoclaved prior to the start of the experiment, and hence, no other biological activity was present.Similar behavior was described by Blánquez et al. [36], who claimed that T. versicolor, under nutrient limitation, can produce laccase while no growth is observed.In fact, no growth may benefit HTL-AP fungal  Note: within each column, means that are followed by the same letter are not significantly different (p < 0.05).
treatment when the goal is to increase the fertilizer value, rather than increase fungal biomass.For instance, excessive fungal biomass on the reactor's walls would be avoided, which is known to be a problem in larger bioreactors [36].In addition, the laccase enzyme released by the fungus may assist in removing potential toxic compounds present in the wastewater as it is known to degrade complex organics [28].Thus, the absence of increased T. versicolor biomass when cultivated in HTL-AP may be advantageous to this wastewater treatment for further use in hydroponic systems, and laccase production may reduce the wastewater toxicity.
Hydroponically grown plants uptake nitrogen as ions, i.e., NH 4 + and NO 3 − [9].In these terms, measurements were taken to monitor any changes in these nitrogen ions during fungal cultivation.These data are important for two reasons: to evaluate whether T. versicolor can increase inorganic nitrogen in HTL-AP, and how long fungal cultivation should be performed to maximize concentrations of these nitrogen forms.There is a gradual increase in NH 3 /NH 4 + -N (Fig. 1, b).No changes in the negative controls for NH 3 /NH 4 + -N concentrations were observed, indicating that the increase in NH 3 /NH 4 + concentration were solely attributed to the presence of T. versicolor.
The time points of 168 and 214 h presented the highest values measured.They are significantly higher than the others but not significantly different from each other, according to the Kruskal-Wallis test (Table 2).After two days of fungal cultivation, NH 3 /NH 4

+
-N increased from 4.04 mg/L to 12.30 mg/L, which aligns with the increase in pH during the same period (Fig. 1, b).HTL-AP pH after two days of fungal cultivation reached 7.52 and remained high during the rest of the days.Depending on the substrate C:N ratio, fungi can metabolize low molecular weight nitrogen organics and release NH 3 /NH 4 + in this process, known as ammonification [20,21].It is likely that T. versicolor performed ammonification, metabolizing short nitrogen organic chains and releasing the excess of nitrogen in the wastewater as NH + are in equilibrium, and their concentration depends on the solution pH [37].Considering the pH of HTL-AP after fungal cultivation, and room temperature, it is possible to say that most of the NH 3 /NH 4 + -N in the wastewater after treatment is in its ionic (NH 4

+
) form, which is preferred by hydroponically grown plants, since pKa is higher than pH (pKa = 9.25, pH = 7.52-8.12[38]).An increase in NH 4 + during T. versicolor wastewater treatment has been reported by Dalecka et al. [39] when treating non-sterile municipal wastewater.They observed an increase in NH 4 + when wastewater pH was not adjusted (pH = 7.5-7.6),and no NH 4 + changes occurred when wastewater pH was adjusted to pH = 5.5.Similarly, Hultberg and Bodin [40] stated that no changes happened in NH 4 + concentration when treating brewery wastewater with pH = 6.6.NO 3 − -N results are found in Fig. 2 (a).An increase in NO 3 − -N is observed over the first 3 days, peaking at 30.67 mg/L.This shows a remarkable increase of 17 times in NO 3 − concentration compared to the initial concentration in 5 % HTL-AP.No changes in the negative controls for NO 3 − -N concentrations were observed, implying that the increase in NO 3 − concentration was solely due to the presence of T. versicolor.The time points of 72 and 94 h had a significantly higher NO 3 − -N than all the others, but they were not statistically significant from each other (Table 2).Nitrification has been reported in different filamentous fungi [41].However, since no decrease in -N or nitrogen-organics by these microbes, which includes recalcitrant compounds and small chains, may result in NO 3 − generation; however, not much information exists about specific mechanisms responsible for this in fungi [42].In these terms, it is speculated that T. versicolor may have performed nitrification using nitrogen organics over the first three days of cultivation.On the fourth day of fungal treatment, a decline in NO 3 − -N concentration is observed.is noticed that after the third day, the increase in NH 3 /NH 4 + -N concentration seems to reflect the decrease in NO 3 − -N.Some fungi may uptake NO 3 − as a nitrogen source for growth and release NH 4 + [27,43].However, specific enzymes, i.e., nitrate reductases, are necessary for fungal NO 3 − uptake and further NH 4 + generation [44]; NO 3 − -N assimilation by fungi may only take place in the absence of other preferred nitrogen sources [45].Although it seems T. versicolor did not prefer NH 3 /NH 4 + -N as its primary nitrogen source, it is not possible to confirm whether this fungus produced the required enzymes to assimilate NO 3 − .In addition, as no growth could be observed (Fig. 2), there is no evidence that NO 3 − -N assimilation was used for growth.Still, it is hypothesized that the fungus consumed NO 3 − in the absence of another preferred nitrogen source.The temporal study aimed to evaluate important parameters during HTL-AP fungal treatment; the T. versicolor cultivation period was one of them.Hydroponic plants can absorb both NH 4 + and NO 3 − as nutrients, but excess of the former is known to harm them [9,46].
In this study, NO 3 − -N peaks on the third day of fungal cultivation (30.67 mg/L); on the same day, the pH reached 7.49 and the NH 3 /NH 4 + -N concentration was 30.20 mg/L, 8 times higher than the starting concentration.In addition, an increase in NO 3 − concentration may benefit the future use of the treated wastewater as a fertilizer source.The NO 3 − /NH 4 + ratio has been reported to impact hydroponic systems; an increase in the ratio tends to improve hydroponic plant growth [47,48].Nitrifying bacteria could be used to increase the NO 3 − /NH 4 + ratio of the treated HTL-AP.Nitrifying bacteria from Nitrosomonas and Nitrobacter genus can assist NH 4 + conversion into NO 3 − [9,49].Bacterial nitrification was used by Jesse and Davidson [7] to increase NO 3 − and nitrite (NO 2 − ) in 5 % HTL-AP.An increase of only 1.75 mg/L of NO 3 − + NO 2 − was achieved, but HTL-AP toxicity or insufficient nitrification time may have directly impacted this small increase.Here, fungal treatment for three days increased the NO 3 − -N concentration by 28.83 mg/L.In addition, fungi possess the ability to remove pollutants, such as aromatic/nitrogen organics and heavy metals, that are known to be responsible for HTL-AP toxicity [4,28], which may improve nitrifying bacteria performance after T. versicolor treatment.Finally, three days of HTL-AP fungal treatment appears to provide the highest NO 3 − -N concentration; subsequently adding nitrifying bacteria could then further increase the NO 3 − concentration by converting NH 4 + to NO 3 − .
The COD analysis relates to the number of organics and the level of pollution in the liquid [50].The COD analysis is shown in Fig. 3 and Table 2.In the first 24 h, T. versicolor removed 51.33 % of COD.This reduction was expected and has been reported by other studies.Cerrone et al. [51] demonstrated a 72 % COD removal when using this fungus to treat olive-washing wastewater.Similarly, Hultberg and Bodin [40] achieved a 67.1 % COD removal when treating brewery wastewater for 13 days.Boujelben et al. [30] reported COD removal from tannery wastewater by T. versicolor alive (31.2-45 %) and dead (19 %), showing the fungus' capacity to adsorb organic pollutants into its mycelium structure.Nonetheless, after two days of treatment, COD increased and remained constant for several days.The time points from 72 to 214 h presented significantly higher COD values than of times points 24 and 46 h (Table 2).Boujelben et al. [30] reported an increase in COD after 7 days of fungal cultivation.They claimed this increase might be due to a lack of nutrients and the production of enzymes or metabolites by the fungus.In addition, constant COD has also been reported in the literature when treating urban wastewater by Cruz-Morató et al. [35].It should be noted that the rise in COD may affect plant growth and could be linked to the formation of byproducts, such as enzymes and metabolites, during fungal treatment.Further research is essential to thoroughly assess these effects.Therefore, T. versicolor can reduce 5 % HTL-AP COD, but not continuously; over time, there was an increase in COD that may be associated with products generated by the fungus.
Submerged cultivation of filamentous fungi has been extensively studied in the literature and integrated into industrial practices [52].Various types of bioreactors have been investigated for filamentous fungi cultivation, such as stirred tanks, bubble-column, packed, and fluidized-bed reactors [52].For instance, promising results have been achieved when using fluidized bed bioreactors to degrade pharmaceuticals by T. versicolor for wastewater treatment [53,54].In this study, three days of T. versicolor HTL-AP treatment showed promising results in enhancing NO  determine optimal performance conditions for the fungus and assess the feasibility of this treatment when scaling up.Additionally, HTL-AP composition can change depending on the HTL feedstock [55], which may alter fungal performance during treatment.Evaluating the impact of different HTL-AP compositions on fungal treatment will also be crucial to assessing the feasibility of employing T. versicolor in this context.Thus, such investigations are essential for the successful implementation of this biotechnological approach.

Wastewater nitrifying experiment
The addition of nitrifying bacteria to HTL-AP after fungal treatment may be beneficial to assist with NH 4 + conversion into NO 3 − , as previously discussed.During nitrification, ammonia-oxidizing bacteria convert NH 3 to NO 2 − , which is further converted to NO 3 − by nitrate-oxidizing bacteria [56].An attempt to apply nitrifying bacteria to increase NO 3 − concentration in 5 % HTL-AP from swine manure has been reported; however, only a 1.75 mg/L increase in NO 3 − + NO 2 − concentration was observed [7].In contrast, we added nitrifying bacteria to the HTL-AP after fungal treatment and compared the outcomes with those obtained from HTL-AP treated with the isolated microbes (Fig. 4).HTL-AP samples inoculated with Trametes versicolor resulted in a noticeable NH 3 /NH 4 + -N concentration increase (Fig. 4), which aligns with results from section 3.1.Bacteria and fungus (B + Tv-5HTL-AP) and fungus (Tv-5HTL-AP) treatments presented values of NO 3 − -N and NH 3 /NH 4 + -N significantly higher than bacteria treatment (B-5HTL-AP) and no treatment (5HTL-AP) (Table 3).An approximately five-times increase in NH 3 /NH 4 + -N was found for samples containing the fungus, with (B + Tv-5HTL-AP) or without (Tv-5HTL-AP) bacteria addition, compared to the control (5HTL-AP).Once again, it is hypothesized that the fungus produced NH 3 /NH 4 + through ammonification.In contrast, the sample only inoculated with bacteria (B-5HTL-AP) decreased the NH 3 /NH 4 + -N concentration 3.7-times compared to the control, which may indicate the microbes have performed nitrification.This claim agrees with the increase in NO 3 − -N for the sample containing only bacteria in 5 % HTL-AP (B-HTL-AP).The concentration of NO 3 − -N increased in all treated samples.The results from samples containing bacteria, fungus and the combination of both resulted in NO 3 − -N concentration increase of 2, 8 and 17 times, respectively, compared to the control.Compared to the fungal treatment (Tv-5HTL-AP), a two-times increase was observed when nitrifying bacteria were added to the treatment (B + Tv-5HTL-AP).These results suggest that the addition of bacteria has a beneficial impact on the treatment for the specific objectives of this study.Thus, it is likely that the fungus is performing ammonification, releasing NH 3 /NH 4 + during the treatment, while bacteria are taking up this inorganic nitrogen and converting it to  Note: within each column, means that are followed by the same letter are not significantly different (p < 0.05).Legend: B + Tv-5HTL-APfungal treatment with nitrifying bacteria addition, Tv-5HTL-APfungal treatment, B-5HTL-AP -HTL-AP treated with only nitrifying bacteria, and 5HTL-AP -negative control.

Conclusion
HTL-AP valorization will benefit the overall value and efficiency HTL process.The use of this wastewater stream as a fertilizer is possible, but requires increasing inorganic nitrogen availability.Here, we demonstrated that a 5 % HTL-AP solution treated with the white-rot fungus T. versicolor for 3 days increased NO 3 − -N concentration from 1.84 mg/L to 30.67 mg/L, and NH 3 /NH 4 + -N from 4.04 mg/L to 32.20 mg/L, representing an increase of almost 17-and 8-times, respectively.It is also worth noting that the introduction of nitrifying bacteria into the 3-day fungal treatment resulted in a two-fold increase of NO 3 − -N compared to fungal treatment alone.Thus, T. versicolor is a great candidate to increase inorganic nitrogen in 5 % HTL-AP; nonetheless, more research is needed to assess the wastewater toxicity and growth of hydroponic plants after the fungal treatment.

Fig. 1 .
Fig. 1.Responses of T. versicolor treatment compared to the negative control: (a) Fungal dry weight and laccase activity and (b) NH 3 /NH 4 + -N and pH measured during T. versicolor cultivation in 5 % HTL-AP over time.

3 /NH 4 +; 4 +-
this might explain the reason for the NH 3 /NH 4 + -N increase over time.It is also possible that T. versicolor may prefer other sources of nitrogen when cultivated in wastewater rather than NH 3 /NH 4 + nitrogen.As a result, NH 4 + produced during ammonification would not be used by the fungus, which may explain the increase in NH 3 /NH N in the samples (Fig. 1, b).NH 3 /NH 4 + -N measured during the experiments accounts for both NH 3 and its ionic form (NH 4 + ); NH 3 /NH 4

Fig. 2 .
Fig. 2. (a) NO 3 − -N and (b) NO 3 − -N and NH 3 /NH 4 + -N measured during T. versicolor cultivation in 5 % HTL-AP over time; average represents the average between NO 3 − -N and NH 3 /NH 4 + -N concentration at the same time; NO 3 -N control refers to the negative control.

Fig. 2 (
b) shows that this decline occurred simultaneously with the rise in NH 3 /NH 4 + -N.Interestingly, adding the average between NO 3 − -N and NH 3 /NH 4 + -N concentrations at each specific time to Fig. 2 (b), it

3 −
-N and NH 3 /NH 4 + -N concentrations in HTL-AP for future use as fertilizer on a bench scale.Future research should evaluate T. versicolor HTL-AP bioremediation using different bioreactor configurations to

Fig. 3 .
Fig. 3. COD measured during T. versicolor cultivation in 5 % HTL-AP along the time compared to the negative control.

Table 2 NO 3
− -N, NH 3 /NH 4 + -N, and COD concentrations along fungal treatment time with their respective statistical analysis (Tukey HSD or Kruskal-Wallis test).